A granulocyte colony-stimulating factor (GCSF), also known as pluripoietin, acts in hematopoiesis by controlling the production, differentiation, and function of the granulocytes that comprises 70% of white blood cells. The growth factor is secreted predominantly by macrophages, fibroblasts, and endothelials with several inflammatory stimuli such as interleukin-1β, tumor necrosis factor-alpha, and lipopolysaccharide. Clinically, human GCSF (hGCSF) has been approved for treatment of neutropenia, a disorder characterized by an extremely low number of neutrophils in blood. Usually cancer patients suffer from this leukopenia after radiotherapy or chemotherapy. Besides, GCSF has also expressed the neuroprotective property. Accordingly, the protein has been applied as a protective agent to mouse models of various neurodegenerative diseases, such as amyotrophic lateral sclerosis.
Initially the production of hGCSF was from a tumor cell line that continuously secreted hGCSF. When expressed in the methylotrophic yeast, Pichia Pastoris, the hormones were secreted in the soluble form but highly aggregated so that they had to be solubilized using high concentrations of denaturants such as guanidine hydrochloride or urea and subsequently refolded by removal of denaturants to be purified as the biologically active form. Many expression studies using bacteria such as Escherichia coli (E. coli) produced also inclusion bodies that is aggregation of overexpressed proteins in the cytoplasm. In many cases, the overall yield of biologically active protein from inclusion body is low. Alternatively, the protein was secreted to periplasm of E. coli, but the yield can be also low.
Although Korean Patent No. 2012-0057416 discloses polynucleotide for producing human G-CSF protein and a use thereof, there is no description about a tagging system such as a water-soluble expression of hGCSF and a hPDIb′a′ domain for its separation according to an aspect of the inventive concept.